Influenza viruses (type A, B, and C) are the causative agents of respiratory infections in humans and other mammalian and avian species. Unique biological and epidemiological characteristics in the virus life cycle drive a rapid antigenic evolution and continuous emergence of new virus strains, particularly of the type A virus. This continuous emergence of antigenic variants allows for seasonal influenza epidemics and at irregular intervals global pandemics. Annual influenza epidemics are associated with approximately 36,000 deaths in the United States alone and more than 250,000 fatalities worldwide each year while causing enormous health and economical burdens. Pandemic influenza poses a serious threat to human health and such global events as the 1918 pandemic caused an estimated 50 millions death and devastating socio-economical effects.
Influenza viruses have a single stranded, segmented RNA genome and are members of the Orthomyxoviridae family. Influenza A viruses display two major glycoproteins on the surface of the virion particle, the hemagglutinin (HA) and the neuraminidase (NA) and based on the antigenic properties of these two molecules they are classified into 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9). The predominant immune response following influenza infection targets the surface glycoprotein HA and NA. Antibodies against the HA are able to block virus binding to the cell surface receptor and prevent virus entry and infection. Over time, however, this response is rendered ineffective and unable to protect against emerging antigenic variants resulting from antigenic drift (accumulation of mutations) or antigenic shift (swapping segment by reassortment).
Currently, one the most effective intervention for the control of seasonal or pandemic influenza disease is prophylactic vaccination. This practice requires that susceptible subjects receive annual doses of vaccine formulated with the most recent human viral isolates. Such vaccines are usually prepared from individual viruses, each one grown in embryonated chicken eggs or cultured cells and then blended in the final vaccine formulation in order to provide a broader coverage. Due to the rapid antigenic variation of the influenza virus, vaccines have to be periodically reformulated to incorporate emerging antigenic variants in order to maintain vaccine protective efficacy.
Vaccine effectiveness, therefore, depends upon the degree of antigenic similarity between the vaccine and virus strains circulating in humans. Furthermore, a certain proportion of vaccinated individuals, including the elderly, fail to develop protective immunity with the current vaccines.
A recent study performed with a phage-display antibody library has identified broadly neutralizing antibodies which are able to block infection across a broad spectrum of influenza virus subtypes (Jianhua Sui et. al. (2009) Nat. Structural & Mol. Biol. 16:265-273.) The ability of these antibodies to neutralize a broad spectrum of virus results from reacting with a sub-dominant and highly conserved epitope present in the stem region of the HA molecule, blocking infection by inhibiting membrane fusion rather than preventing receptor binding which is the predominant neutralizing mechanism induced by virus infection or vaccination. Design of a vaccine that displays these subdominant and highly conserved epitopes will elicit broadly neutralizing immune responses able to protect against a broad spectrum of influenza subtypes.
In order to produce the viral components needed to formulate the current vaccines, selected viruses are grown by infecting embryonated chicken eggs or cultured cells. This process depends upon the virus's ability to attach via the HA molecule to the sialic acid containing receptor on the cell surface, penetration, virus replication and isolation of viral progeny from the fluids in the egg or cultured cells. Truncation, re-engineering or remodeling of the viral HA would eliminate receptor binding and preclude virus replication and the production of such a vaccine.
Influenza VLPs are produced by the simultaneous expression of 2, 3 or 4 viral genes (M1, M2, HA and NA) with M1, M2, or (analogous matrix proteins) HA and NA being the main and essential components of the structure. See, e.g., U.S. Patent Publication Nos. 20080031895 and 20090022762. The DNA sequences encoding these genes are simultaneously or sequentially transfected into cells, where they are transcribed and translated into their respective proteins that self-assemble into virus-like particles. In contrast to viruses, modifications of the regions of the HA used for receptor binding and virus penetration do not interfere with the production, assembly or release of VLPs from the cells. That is, VLP production allows for the deletion and remodeling of major portions of HA bearing immunodominant and genetically variable regions of the molecule whereas the virus replication method does not allow for these modifications. Similar modification can also be introduced on the NA molecules, which is also displayed on the surface of the VLP vaccine.
However, there remains a need for influenza vaccine compositions and methods of making and using such compositions.